Part D: Lecture 32 Flashcards
Electrophoresis (def.)
the separation of charged molecules using an electric field
Gel material does what?
slows down migration of DNA
2 types of gel material
agarose
acrylamide
agarose separates pieces of ____
dsDNA > 100 bp
acrylamide separates pieces of ____
dsDNA < 1 kb
ssDNA
gel material preference
agarose gel (“cheap and edible”)
acrylamide gel problems
expensive and poisonous
Gel apparatus used
Slab gel in a buffer between negative and positive electrode
buffer function in gel electrophoresis
conducts charge between electrode
DNA mix for gel electrophoresis into well
DNA molecules + glycerol + loading/tracking dye
glycerol function in DNA mix
DNA falls into well
loading/tracking dye (dark blue) function
allows us to see wells are loaded
shorter linear pieces of DNA moves _____
faster
longer linear pieces of DNA moves _____
slower
Band (def. gel electrophoresis)
where the DNA ends up
Lane (def. gel electrophoresis)
the path that DNA move
charge of loading/tracking dye
negative charge (same of DNA sample)
visualizing DNA in gel electrophoresis
-Ethidium bromide
-RedSafe
EtBR +DNA/RNA +UV = ___
orange fluorescence
RedSafe +DNA/RNA +UV = ____
green fluorescence
Molecular weight markers (def.)
dsDNA fragments of known sizes and amounts
restriction map (def.)
map of where RE cuts on DNA
restriction fragments (def.)
DNA molecules produced by restriction digest
Order of bands and restriction fragments
order of bands ≠ order of restriction fragments
The fastest bands are ____ (appearance) because the pieces are ___
fainter
shorter (less Etbr in DNA)
Determining the length of DNA procedure
- Run a gel with unknown DNA in one lane + MOL wt markers in another lane
- Take a photo (black and white)
- Measure distance each band travelled from well
4.Sheet of semi-log graph paper (STRAIGHT LINE) - Plot distance of mol wt markers
- Estimate size of unk band
Gel purifying DNA function
an easy way to obtain specific DNA molecules from a diverse collection of DNA
Gel purifying DNA method
1)run DNA in a agarose gel
2)cut out the band and separate the DNA from the agarose + remove agarose with gel extraction kit
Confirming integrity of DNA procedure
1) genomic DNA isolation protocol
2)load on gel directly
2)digest with RE another load on gel
speed that DNA > 20 kb runs ___
as a single band
RNA contamination
smear at the bottom
protein contamination
thick band at the top
